Mutations in structural components of the nuclear lamina cause a spectrum of human diseases known as laminopathies. Specific mechanisms for how disruption of the lamina generates cellular phenotypes have not been elucidated. This proposal tests a new hypothesis for how mutation of the gene encoding lamin A gives rise to cellular phenotypes in the premature aging disease, Hutchinson-Gilford Progeria Syndrome (HGPS). The proposal is based on data showing (i) the nucleocytoplasmic Ran gradient is disrupted in HGPS patient cells and this can be recapitulated in naive cells; (ii) Ran gradient disruption causes mis-localization of the SUMOylation enzyme Ubc9 from the nucleus to the cytoplasm; (iii) Reducing the levels of reactive oxygen species, which are elevated in HGPS cells, restores nuclear localization of the RanGTPase and Ubc9. The overall hypothesis is that constitutive anchoring of pre-lamin A to the nuclear membrane generates reactive oxygen species that inhibit the Ran GTPase System, disrupt the Ran gradient, relocalize Ubc9 to the cytoplasm, and induce cellular phenotypes in Progeria. Thus, defects in the nuclear lamina are transduced into cellular phenotypes, at least in part, via changes in the nuclear transport machinery. In Aim 1 we will determine the source and mechanism of reactive oxygen species induction by Progerin. In Aim 2 we test the hypothesis that the Ran GTPase System acts as a sensor for pre-lamin A- induced oxidative stress. In Aim 3 we test the hypothesis that the SUMO conjugating enzyme Ubc9 functions as an effector of oxidative stress by responding to the Ran gradient and modulating gene expression. Our studies bring a new perspective to the Progeria field, which includes a signaling and nuclear transport-based based framework to help understand how disease phenotypes are generated at the cellular level. PUBLIC HEALTH RELEVANCE: The nucleus is a membrane-enclosed organelle that contains DNA, the genetic material that provides the blueprint for making proteins and determining cellular function. The structure and function of the nucleus depends on an elaborate scaffold termed the nuclear lamina. Mutations in the proteins that form the nuclear lamina cause human diseases known as laminopathies. The goal of our research is to determine how mutation of lamin A (a major component of the nuclear lamina) gives rise to the premature aging disease, Hutchinson-Gilford Progeria Syndrome.